Thermo Fisher Scientific Dynabeads Protein A Immunoprecipitation Kit User guide

Brand: Thermo Fisher Scientific

Model: Dynabeads Protein A Immunoprecipitation Kit

Type: User guide

Kit contents

Component Volume

Dynabeads™ Protein A 2 mL

Ab Binding & Washing Buffer 16 mL

Washing Buffer 28 mL

Elution Buffer 1 mL

Dynabeads™ Protein A kit contains

sufcient reagents for 40 reactions. The

magnetic beads are at a concentration of

30mg/mL in phosphate buffered saline

(PBS), pH 7.4, with 0.01% Tween™-20

and 0.09% sodium azide as a

preservative.

Caution: Sodium azide may react with

lead and copper plumbing to form

highly explosive metal azides.

Note: Read the Safety Data Sheets

(SDSs) for additional information on

buffers.

Product description

The kit contains Dynabeads™ Protein

A beads, as well as buffers for

binding, washing, and elution steps

for immunoprecipitation of proteins,

protein complexes, protein-nucleic acid

complexes, and other antigens.

Dynabeads™ Protein A

Immunoprecipitation Kit

For research use only. Not for use in diagnostic procedures.

Catalog No. 10006D Store at 2˚C to 8˚C

Publication No. MAN0017347 Rev. A.0

Antibodies (Ab) are added to the

Dynabeads™ Protein A, and bind to the

magnetic beads via their Fc-region during

a short incubation. The tube is placed on a

magnet, and the beads adhere to the side

of the tube facing the magnet, allowing

easy removal of the supernatant.

The bead-bound Ab is then used for

immunoprecipitation of the target antigen

(Ag). Bound material is easily collected

utilizing the unique magnetic properties of

the Dynabeads™ magnetic beads.

The Dynabeads™ Protein G kit can also be

used as part of an automated workow

on the KingFisher™ platform. Protocols for

the KingFisher™ Duo Prime System (up to

24 samples/run) and KingFisher™ Flex™

System (up to 96 samples/run), go to

thermosher.com/kingsher.

Required materials

• DynaMag™ Magnet (See

thermosher.com/magnets for

recommendations).

• Sample mixer allowing tilting and

rotation of tubes (e.g., HulaMixer™

Sample Mixer).

General guidelines

• Dynabeads™ Protein A has a binding

capacity of ~8 µg of human IgG

per mg of beads. The amount

of Ab captured depends on the

concentration of beads and Ab in the

starting sample, as well as the type of

immunoglobulin being bound.

• The choice of primary antibody is the

most important factor for successful

target Ag capture. Some antibodies

exhibit reduced Ag-binding efciency

for immunoprecipitation, even though

good results are seen with other

immunological assays. The afnity of

different antibody types to Protein A is

shown in Table 1.

• For low-afnity antibodies or samples with low antigen concentration, pre-

incubate the sample and antibody (indirect technique) before bead capture

to improve binding kinetics for the antibody and minimize non-specic

binding. This approach is also recommended when working with protein/

nucleic acid complexes, e.g., ChIP.

• Due to fast binding kinetics, an antibody only needs to be incubated with the

Dynabeads™ magnetic beads for 10 minutes for sufcient antibody capture.

• Increase incubation time of the magnetic bead-Ab complex with the target

antigen to 20–120 minutes to increase yield for low afnity antibodies or

samples with low antigen concentration, although this may lead to increased

non-specic binding.

• For sensitive proteins and phosphorylation studies, perform the isolation

protocol and elution at 2–8°C, to avoid protein complex dissociation and

minimize enzymatic activity.

Protocol

The protocol is a general guideline for immunoprecipitation. Optimization may

be required for each antibody (Ab) and target antigen (Ag). The protocol uses

50 µL of Dynabeads™ Protein A, but this may be scaled up or down as required.

Prepare Dynabeads™ magnetic beads

1. Resuspend Dynabeads™ magnetic beads in the vial (vortex >30 sec or tilt and

rotate 5 minutes).

2. Transfer 50 µL (1.5 mg) Dynabeads™ magnetic beads to a tube.

3. Place the tube on the magnet to separate the beads from the solution, and

remove the supernatant.

4. Remove the tube from the magnet.

5. Proceed directly to "Bind antibody".

Bind antibody

1. Add your antibody (typically 1–10 μg) diluted in 200 µL of Ab Binding and

Washing Buffer to the magnetic beads from step 4 in “Prepare Dynabeads™

magnetic beads”. The optimal amount of Ab depends upon the individual

Ab used.

2. Incubate with rotation for 10 minutes at room temperature.

3. Place the tube on the magnet and remove the supernatant.

4. Remove the tube from the magnet and resuspend the magnetic bead-

Ab complex in 200µL Ab Binding and Washing Buffer. Wash by gentle

pipetting.

Note: Ab-conjugated magnetic beads can be stored in Ab Binding and

Washing Buffer to prevent aggregation.

5. Proceed to “Immunoprecipitate target antigen”.

(Optional) Crosslink antibody

To avoid co-elution of your antibody, crosslink your antibody to the Dynabeads™

magnetic beads before immunoprecipitation. Use the crosslinking reagent

DSS (disuccinimidyl suberate). For further information and procedure, visit

thermosher.com/proteincrosslinking.

Immunoprecipitate target antigen

1. Place the tube (from step 4 of “Bind antibody”) on the magnet and remove

the supernatant.

2. Add your sample containing the antigen (typically 100–1,000 µL) and gently

pipette to resuspend the magnetic bead-Ab complex.

3. Incubate with rotation for 10 minutes at room temperature to allow the

antigen to bind to the magnetic bead-Ab complex.

Note: Depending on the afnity of the antibody, it may be necessary to

increase incubation times for optimal binding.

Figure 1: Principle of immunoprecipitation of

antigen using Dynabeads™ Protein A.

Dynabeads ProteinA

™

Bind antibody

(Crosslink antibody optional)

Immunoprecipitate

target antigen

Elute target antigen

For support visit thermoﬁsher.com/support or

email [email protected]om

thermoﬁsher.com

14 September 2017

Corporate entity: Life Technologies | Carlsbad, CA 92008 USA | Toll Free in USA 1.800.955.6288

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DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,

INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,

INCLUDING YOUR USE OF IT.

Ig origin Afﬁnity for Protein A

Human IgG1,2,4 +++

Human IgD –

Human IgA, E, M +

Human IgG3 +

Mouse IgG1 +

Mouse IgG2, 2b, 3 +++

Mouse IgM +

Rat IgG1 +

Rat IgG2a –

Rat IgG2b –

Rat IgG2c +++

Bovine IgG1 +

Bovine IgG2 +++

Chicken IgY –

Dog IgG +++

Goat IgG1 +

Goat IgG2 +++

Guinea Pig IgG +++

Hamster +

Horse IgG +

Monkey IgG +++

Porcine IgG +++

Rabbit IgG +++

Sheep IgG1 +

Sheep IgG2 +++

Table 1: Binding strength of Protein A to different species of Ig’s and their

subclasses.

4. Place the tube on the magnet. Transfer the supernatant to a clean tube for

further analysis, if desired.

5. Wash the magnetic bead-Ab-Ag complex 3 times using 200 µL Washing

Buffer for each wash. Separate on the magnet between each wash, remove

supernatant, and resuspend by gentle pipetting.

6. Resuspend the magnetic bead-Ab-Ag complex in 100 µL Washing Buffer and

transfer the bead suspension to a clean tube. This is recommended to avoid co-

elution of proteins bound to the tube wall.

Note: To store the immunoprecipitated protein, add Elution Buffer and

NuPAGE™ LDS Sample Buffer, then freeze the magnetic bead-Ab-Ag complex.

For subsequent analysis of the sample, thaw and proceed with step 2 of "Elute

target antigen (Denaturing elution)".

7. Proceed to “Elute target antigen”.

Elute target antigen

Denaturing elution

1. Place the tube containing the magnetic bead-Ab-Ag complex on the magnet

and remove the supernatant.

2. Add 20 µL Elution Buffer and 10 µL of pre-mixed NuPAGE™ LDS Sample

Buffer and NuPAGE Sample Reducing Agent.

3. Gently pipette to resuspend the magnetic bead-Ab-Ag complex.

4. Heat for 10 min at 70ºC.

5. Place the tube on the magnet and load the supernatant/sample onto a gel.

Note: As an alternative, the magnetic bead-Ab-Ag complex can be resuspended in

a sample buffer of your choice (e.g. SDS sample buffer). Follow the recommended

temperatures and heating times for these buffers prior to gel loading.

Non-denaturing elution

1. Place the tube (from step 6 "Immunoprecipitate target antigen") on the magnet

and remove the supernatant.

2. Add 20 µL Elution Buffer and gently pipet to resuspend the magnetic bead-Ab-

Ag complex. Avoid producing foam during resuspension.

3. Incubate with rotation for 2 min at room temperature to dissociate the complex.

4. Place the tube on the magnet and transfer the supernatant containing eluted Ab

and Ag to a clean tube. If the eluted protein is to be used for functional assays

or stored, the pH of the eluate can be adjusted by adding 1 M Tris, pH 7.5.

Description of materials

This product contains Dynabeads™ Protein A for immunoprecipitation.

Dynabeads™ Protein A are uniform, 2.8 μm, superparamagnetic beads with

recombinant Protein A (approximately 45 kDa) covalently coupled to the surface.

Thermo Fisher Scientific Dynabeads Protein A Immunoprecipitation Kit User guide

Brand: Thermo Fisher Scientific

Model: Dynabeads Protein A Immunoprecipitation Kit

Type: User guide

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Thermo Fisher Scientific Dynabeads Protein A Immunoprecipitation Kit User guide

Thermo Fisher Scientific Dynabeads Protein A Immunoprecipitation Kit User guide

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